Project Summary Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (CRISPR- Cas) systems in bacteria and archaea can specifically bind and cut a sequence of DNA in a programmable, RNA-guided manner. For this reason, CRISPR effector nucleases, particularly S. pyogenes Cas9 (SpyCas9) and Acidaminococcus sp. Cas12a (AsCas12a), which cut DNA as single-protein effectors, are promising tools for therapeutic genome editing. These bacterial proteins can be deployed in human nuclei, along with an engineered RNA transcript known as a single guide RNA (sgRNA), to specifically target any DNA sequence in the genome, provided it is complementary to a 20-nt spacer sequence in the sgRNA and contains a short protospacer adjacent motif (PAM) next to the target sequence. Though these CRISPR-Cas tools have many possible future uses, the challenges of controlling off-target cleavage events and editing outcomes of CRISPR- induced DNA breaks must be overcome to realize their therapeutic potential. Improved understanding of CRISPR effectors in live cells would provide insight into strategies for better control of CRISPR-Cas in human nuclei. A live-cell imaging platform, in which cells stably express nuclease- dead SpyCas9 fused to a HaloTag domain, has been successfully used to visualize Cas9 DNA interrogation in live mouse cell nuclei. Using this CRISPR imaging platform as a foundation, I propose to i) construct stable cell lines expressing WT and nuclease-dead variants of SpyCas9 and AsCas12a, fused to a HaloTag domain for imaging ii) compare binding fidelity of SpyCas9 and AsCas12a targeted to orthogonally labeled DNA sequences and iii) image fluorescently labeled WT CRISPR effectors and human repair enzymes to map the sequence and duration of steps processing CRISPR-induced DNA breaks. Specifically, by measuring the lag time between binding of CRISPR effectors and binding of various individual repair enzymes on target DNA, I will spatiotemporally map substrate transfer from CRISPR-Cas to repair. The findings of this study will provide a direct visualization and comparison of Cas9 and Cas12a on-and off- target binding in cells. This study will also identify possible rate-determining steps for homology-directed repair outcomes in processing CRISPR-induced DNA breaks, providing a road map to engineering high-fidelity repair outcomes in Cas9- or Cas12a- edited cells. Finally, this study may identify possible alternative pathways for high-fidelity repair outcomes, which could be useful in treating cancer patients with mutations in BRCA1 and other proteins known to contribute to CRISPR-induced DNA break processing.